Development and validation of UPLC method for WST-1 cell viability assay and its application to MCTT HCE™ eye irritation test for colorful substances.
Joo KM, Kim S, Koo YJ, Lee M, Lee SH, Choi D, Lim KM.Abstract
WST-1 [Water Soluble Tetrazolium-1; 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt)] is widely used in the cell viability assays replacing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). A water-soluble formazan dye (4-[1-(4-Iodophenyl)-5-(4-nitrophenyl)formaz-3-yl]-1,3-benzene disulfonate, disodium salt) is produced from the reduction of WST-1 tetrazolium, of which optical density at 450 nm is measured to evaluate cell viability. Colorful substances may interfere with spectrometric measurement, and a method to specifically detect WST-1 formazan is required. Here, a simple, rapid, sensitive, and specific ultra-performance liquid chromatography coupled to UV detector (UPLC-UV) was developed and validated for the WST-1 formazan. For the application to cell viability assay, the supernatant from WST-1 assay was injected without sample preparation procedure and a single run was completed within 5 min. Chromatographic separation was achieved on BEH C18 column (1.7 μm, 2.1 × 50 mm) using gradient elution with the mobile phase of water and acetonitrile. The standard curves were linear over the concentration range of 2.5-120 μg/mL WST-1 formazan, which encompasses WST-1 formazan concentrations from 2% cell viability to 2 fold of 100% cell viability. The intra- and inter-day precisions were measured to be below 5% and accuracies were within the range of 91.8-104.9%. The validated method was successfully applied to the test of colorful substances in vitro eye irritation test with a human cornea-like epithelium, and in vitro cytotoxicity in HaCaT, human keratinocyte cell line.Keywords
Color interference; Cytotoxicity assay; Method validation; UPLC; WST-1 assay
Me-too validation study for in vitro eye irritation test with 3D-reconstructed human cornea epithelium, MCTT HCE™.
Lim SE, Ha SJ, Jang WH, Jung KM, Jung MS, Yeo KW, Kim JS, Jeong TC, Kang MJ, Kim SY, Lee SH, Ko KY, Kim TS, Park KS, Bae S, Lim KM.Abstract
The need for in vitro eye irritation test replacing in vivo is steadily increasing. The MCTT HCE™ eye irritation test (EIT) using 3D reconstructed human cornea-like epithelium, was developed to identify ocular irritants from non-irritants those that are not requiring classification and labelling for eye irritation. Here, we report the results of me-too validation study, which was conducted to evaluate the reliability and relevance of the MCTT HCETM EIT, according to performance standards (PS) of OECD TG 492. The optimal cutoff to determine irritation in the prediction model was preliminarily established at 45% with the receiver operation characteristics (ROC) curve for 141 reference substances. To demonstrate the reproducibility of within- and between-laboratory (WLR and BLR), a set of 30 PS reference chemicals were tested in three laboratories three times. The WLR and BLR concordance with the binary decision of whether non-irritant or irritant was estimated to be 90-100% and 90%, respectively, and both met the PS requirements. The predictive capacity of the respective laboratories for the 30 reference chemicals were evaluated based on three different estimation methods, and the results were comparable, with sensitivity ranging from 89.6 to 93.3%, the specificity ranging from 62.2 to 66.7%, and the accuracy ranging from 75.9 to 80.0%. Additional test with the new set of 30 PS substances in the revised OECD GD 216 yielded a performance of sensitivity ranging from 92.6-93.3%, the specificity 62.2-66.7% and the accuracy 77.4-80.0%. 95.0% sensitivity, 67.2% specificity, and 83.0% accuracy were obtained for 141 reference substances in total. Furthermore, separate cutoffs for liquids and solids, 35% and 60%, respectively, produced better predictivity, which was established as a final prediction model. Collectively, our study demonstrated that MCTT HCE™ EIT meets the reproducibility and predictivity criteria stated in OECD TG 492 PS.Keywords
Eye irritation test; MCTT HCE™; Me—too validation; Predictive capacity; Reconstructed human cornea-like epithelium, RhCE; Reproducibility
The epithelial zinc transporter ZIP10 epigenetically regulates human epidermal homeostasis by modulating histone acetyltransferase activity
Bin BH, Lee SH, Bhin J, Irié T, Kim S, Seo J, Mishima K, Lee TR, Hwang D, Fukada T, Cho EGAbstract
· BACKGROUND: The skin is the first organ that manifests changes in response to zinc deficiency. However, the molecular mechanism underlying how zinc is involved in skin homeostasis, especially its epigenetic regulation, is largely unknown. · OBJECTIVES: In this study we demonstrate the importance of zinc levels and the zinc transporter ZIP10 in the epigenetic maintenance of human epidermal homeostasis. · METHODS: Adult human skin, including skin appendages, were stained with anti-ZIP10 antibody. Histone acetyltransferase (HAT) activity was assessed after treating human keratinocytes with ZIP10 small interfering (si)RNAs or the zinc chelator TPEN. ZIP10- or HAT-regulated genes were analysed based on limma bioinformatics analysis for keratinocytes treated with ZIP10 siRNAs or a HAT inhibitor, or using a public database for transcription factors. A reconstituted human skin model was used to validate the role of ZIP10 in epidermal differentiation and the functional association between ZIP10 and HAT. · RESULTS: ZIP10 is predominantly expressed in the interfollicular epidermis, epidermal appendages and hair follicles. ZIP10 depletion resulted in epidermal malformations in a reconstituted human skin model via downregulation of the activity of the epigenetic enzyme HAT. This decreased HAT activity, resulting from either ZIP10 depletion or treatment with the zinc chelator TPEN, was readily restored by zinc supplementation. Through bioinformatics analysis for gene sets regulated by knockdown of SLC39A10 (encoding ZIP10) and HAT inhibition, we demonstrated that ZIP10 and HATs were closely linked with the regulation of genes related to epidermal homeostasis, particularly filaggrin and metallothionein. · CONCLUSIONS: Our study suggests that ZIP10-mediated zinc distribution is crucial for epidermal homeostasis via HATs. Therefore, zinc-dependent epigenetic regulation could provide alternatives to maintaining healthy skin or alleviating disorders with skin barrier defects.Keywords
Zinc, ZIP10, human epidermal homeostasis
Nervonoylceramide (C24:1Cer), a lipid biomarker for ocular irritants released from the 3D reconstructed human cornea-like epithelium, MCTT HCE™.
Lee M, Joo KM, Choi S1, Lee SH, Kim SY, Chun YJ, Choi D, Lim KMAbstract
Due to invasive and painful procedures during in vivo rabbit eye irritation test, in vitro alternative methods have been widely investigated. Recently, 3D reconstructed human cornea-like epitheliums (RhCEs) garner a huge attention. RhCEs employ the tissue viability as a primary endpoint to determine ocular irritancy but additional biomarkers may improve its predictive capacity. Here, we explored lipid biomarkers for ocular irritants in MCTT HCE™ RhCE model. Three irritants; sodium lauryl sulfate, benzalkonium chloride and triton X-100 were selected to represent anionic, cationic and non-ionic detergent respectively. After treating MCTT HCE™ with irritants, the alteration of lipids in the treated tissues was examined with Nile Red staining, which revealed the depletion of corneal lipids. We further quantitated the release of ceramides and free fatty acids, major lipid components of cornea, into the medium during the post-treatment incubation, employing a sensitive UPLC-MS/MS method. Among 44 lipid species, nervonoylceramide (C24:1Cer) was found to be released commonly by all three irritants in a concentration-dependent manner. Tests with 10 additional reference substances further supported that C24:1Cer release was significantly correlated with viability. Examination of the genes involved in the biosynthetic pathway for C24:1Cer revealed that stearoylCoA desaturase (SCD) and elongase1 (ELOVL1) were upregulated, suggesting that lipids and related genes may be employed as biomarkers for ocular irritants.Keywords
3D reconstructed human cornea-like epithelium; Eye irritation; Lipidomics; Nervonoylceramide. C24:1 ceramide
Development of a new reconstituted human airway model to assess inhalation toxicity
Seolyeong Kim, Hye Soo Kim, Sun Young Jo, Su-Hyon Lee, Ha Ryong KimAbstract
In 2011, polyhexamethylene guanidine phosphate (PHMG) was used as a disinfectant to prevent growth of microorganisms in humidifiers. However, exposure to PHMG was suspected as a causative agent of lung disease in Korea. Thus, determining the toxicity potential of compounds is of importance for health safety. Currently, there are no adequate predictive in vitro models available. Therefore, this study developed a novel in vitro three-dimensional human airway model (HAM model) to predict toxicity as an alternative to animal testing. Two of microbiocides were treated to HAM model, and after three-hour incubation, the tissues were washed with DPBS. Test chemicals were removed, and values of cell viability, cytokine through ELISA and TEER were measured. By the results, we could predict that PHMG and bleomycin possessed irritant potential on the human airway model. Therefore, HAM model can be used as a good alternative test method for assessing toxicity of chemicals.Keywords
inhalation irritation, Reconstructed human airway model, SoluAirway™ model
Improvement of predictive ability to distinguish skin irritants using reconstructed human epidermis model, KeraSkin™-VM
Hye Soo Kim, Seolyeong Kim, Sun Young Jo and Su-Hyon LeeAbstract
Regarding animal welfare which became a social issue in developed countries, various research substituting animal experiments are actively being proceeded. KeraSkin™-VM (Biosolution Co., Ltd., Korea) is an experimental skin model that possesses similar structure and function with human skin, and can be used to perform skin irritation test (SIT). In this study, we tried to optimize the KeraSkin™-VM SIT method to improve its predictive potency for our aim to register it at OECD TG 439 as a validated method. In order to identify the conditions to improve the predictive potency of original KeraSkin™-VM, several trials were performed with different exposure volumes, exposure periods and chemical application method utilizing borderline chemicals contained in the reference chemicals listed in the OECD TG 439 for in vitro SIT. After modifications of KeraSkin™-VM SIT, we examined its predictability with 20 chemicals used in a validation study of Labcyte EPI-MODEL 24 which is an approved validated reference method (VRM). The predictive potency of the modified KeraSkin™-VM SIT was evaluated through its sensitivity, specificity and overall accuracy, where 100%, 71% and 80% was obtained, respectively. These results meet the acceptance criteria described in OECD TG 439, supporting KeraSkin™-VM as a possible candidate to be a registered validated method.Keywords
Skin irritation test, KeraSkin™, Reproducibility, Predictive capacity, Statistical analysis
Improvement of predictive ability to distinguish skin irritant using reconstructed human epidermis model, KeraSkin™-VM, by changing washing method
Jung Eun Park, Jin-Sik Kim, Seolyeong Kim, Hye Soo Kim, Eun-Joo Kim, Kung-min Lim, Su-Hyun LeeAbstract
Regarding animal welfare which became a social issue with developed countries, various researches substituting animal experiment are actively being proceeded. In this study, we aim to register at OECD TG 439 through the evaluation of experimental skin model KeraSkin™-VM (Biosolution Inc., Korea) having the structure and function like human skin. We tried to meet the OECD TG 439 criteria by optimizing a washing protocol to solve the problems. After test material was applied for 45 minutes, using a squeeze bottle was carried out compared with the conventional washing using a pipette. The cell viability was measured by MTT assay and the cell viability was determined to be non-stimulated based on 50% or less. For the 10 reference chemicals test, the specificity 83%, sensitivity 100%. In both cases, 1-bromohexane(1-BHX) was proved to be an irritant and 91% accuracy was achieved, reaching the OECD accuracy standard.Keywords
Reconstructed human skin model, Skin irritation test, KeraSkin™
Investigation of dermal toxicity of ionic liquids in monolayer-cultured skin cells and 3D econstructed human skin models
Hwang JH, Park H, Choi DW, Nam KT, Lim KMAbstract
In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced blockage of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals.Keywords
Human foreskin keratinocyte; Immortalization; Three-dimensional skin culture; Toxicity assessment
PAL-12, a new anti-aging hexa-peptoid, inhibits UVB-induced photoaging in human dermal fibroblasts and 3D reconstructed human full skin model, Keraskin-FT™
Song D, Park H, Lee SH, Kim MJ, Kim EJ, Lim KMAbstract
Peptoids are a class of peptidomimetics whose pharmacological activities are widely investigated owing to their remarkable biological stability. However, the utilities of peptoids as cosmetic functional ingredients have not been fully explored. Here, we investigated anti-aging effects of PAL-12, a new hexa-peptoid, on UVB-induced photoaging in human dermal fibroblasts (HDFs) and a 3D reconstituted human full skin model, Keraskin-FT™. PAL-12 suppressed matrix metalloproteinase-1 (MMP-1) expression induced by UVB irradiation along with the attenuation of MMP-1 secretion as determined by ELISA assay. Interestingly PAL-12 slightly enhanced the expression levels of collagen-1 and fibronectin-1 in HDFs or Keraskin-FT™. In addition, PAL-12 prevented the decrease of cell viability following UVB irradiation. However, PAL-12 failed to affect ROS generation, cell necrosis and apoptosis significantly. Instead, PAL-12 suppressed UVB-induced activation of epidermal growth factor receptors (EGFR), extracellular signal-regulated kinase (ERK) and c-Jun, which may resulted in the attenuation of AP-1-promoted MMP-1 expression. Collectively, these results suggest that PAL-12 might be a novel cosmetic ingredient effective against UVB-induced skin photoaging.Keywords
Epidermal growth factor receptor (EGFR); Keraskin-FT™; Matrix metalloproteinases-1; Peptoid; Photoaging; UVB
Establishment of the new in vitro nasal epithelial model (SoluAir™) for alternative toxicity test
Hye Soo Kim, Seolyeong Kim, Jung Eun Park, Ji-Hye Jang, Su-Hyon LeeAbstract
Human 3D airway model is a promising model for safety and efficacy evaluation of compounds targeting the airway. Therefore, the present study attempted to establish the new in-vitro nasal epithelial model (SoluAir™) for alternative toxicity test. Human nasal epithelial (HNE) cells were seeded onto 12 mm cell culture insert, cultured for 7 days of submerged condition and following 7 days of air-liquid interface condition. Fully differentiated cell sheets were compared with intact human nasal epithelium by H&E and immunohistochemical staining. We confirmed primary cultured HNE cells showed cobblestone-like morphology and well differentiated ciliogenesis. Furthermore, after long term period of ALI(30 days), SoluAir™ showed an increased cilia and maturation of goblet cells. These results suggest that SoluAir™ is suitable for alternative toxicity test owing to its similarity with nasal epithelium.Keywords
reconstructed human Airway, SoluAirwayTM, Alternative toxicity test
Test the delivery temperature for stability of 3D reconstructed human corneal epithelium tissue model, SoluEye™
Seolyeong Kim, Hye Soo Kim, Jung Eun Park, Ji-Hye Jang, Kung-min Lim, Su-Hyon LeeAbstract
Many efforts are being made to develop new alternative in vitro test methods for the eye irritation test. The SoluEyeTM(MCTT HCE™) is a new 3D reconstructed human cornea epithelium model for alternative to animal testing on drugs, cosmetics and raw materials. The need for non-animal ocular test method in neighboring Asian countries, including Japan and China, is increasing. Therefore, we investigated the delivery temperature for the stability of SouEye™ (MCTT HCE™) model. We conducted endpoint analysis including transepithelial electrical resistance (TEER), effective time-50(ET-50) for Triton X-100, Histology using the SoluEye™(MCTT HCE™). By increasing the time for shipment from 12hours to 72hours, SoluEye was confirmed that it was stable at 4 degrees and 18 degrees for 3days.Keywords
reconstructed human Cornea model, MCTT HCE™, SoluEye™, Delivery test
Alternatives to In Vivo Draize Rabbit Eye and Skin Irritation Tests with a Focus on 3D Reconstructed Human Cornea-Like Epithelium and Epidermis Models
Lee M, Hwang JH, Lim KMAbstract
Human eyes and skin are frequently exposed to chemicals accidentally or on purpose due to their external location. Therefore, chemicals are required to undergo the evaluation of the ocular and dermal irritancy for their safe handling and use before release into the market. Draize rabbit eye and skin irritation test developed in 1944, has been a gold standard test which was enlisted as OECD TG 404 and OECD TG 405 but it has been criticized with respect to animal welfare due to invasive and cruel procedure. To replace it, diverse alternatives have been developed: (i) For Draize eye irritation test, organotypic assay, in vitro cytotoxicity-based method, in chemico tests, in silico prediction model, and 3D reconstructed human cornea-like epithelium (RhCE); (ii) For Draize skin irritation test, in vitro cytotoxicity-based cell model, and 3D reconstructed human epidermis models (RhE). Of these, RhCE and RhE models are getting spotlight as a promising alternative with a wide applicability domain covering cosmetics and personal care products. In this review, we overviewed the current alternatives to Draize test with a focus on 3D human epithelium models to provide an insight into advancing and widening their utility.Keywords
3D reconstructed human cornea-like epithelium (RhCE) models; 3D reconstructed human epithelium (RhE) models; Alternative to animal tests; Eye irritation; Skin irritation
Evaluation of ocular irritancy of coal-tar dyes used in cosmetics employing reconstructed human cornea-like epithelium and short time exposure tests
Lee M, Nam KT, Kim J, Lim SE, Yeon SH, Lee B, Lee JY, Lim KMAbstract
Coal-tar dyes in cosmetics may elicit adverse effects in the skin and eyes. Countries, like the US, have banned the use of coal-tar dyes in cosmetics for the eye area due to the potential for ocular irritation. We evaluated the eye irritation potential of 15 coal-tar dyes permitted as cosmetic ingredients in reconstructed human cornea-like epithelium (RhCEs [EpiOcular™ and MCTT HCE™]) tests and the short time exposure (STE) test. Eosin YS, phloxine B, tetrachlorotetrabromofluorescein, and tetrabromofluorescein were identified as irritants in RhCEs; dibromofluorescein and uranine yielded discrepant results. STE enabled further classification in accordance with the UN Globally Harmonized System of Classification and Labelling of Chemicals, as follows: eosin YS as Cat 2; phloxine B, Cat 1; and tetrachlorotetrabromofluorescein and tetrabromofluorescein, Cat 1/2. STE indicated dibromofluorescein (irritant in EpiOcular™) and uranine (irritant in MCTT HCE™) as No Cat, resulting in the classification of "No prediction can be made." based on bottom-up approach with each model. These results demonstrated that in vitro eye irritation tests can be utilized to evaluate the potential ocular irritancy of cosmetic ingredients and provide significant evidence with which to determine whether precautions should be given for the use of coal-tar dyes in cosmetics or other substances applied to the eye area.Keywords
3D reconstructed human cornea-like epithelium; Coal tar dyes; Eye irritation; RhCE; STE; Short time exposure test; UN GHS categorization
Requirement of zinc transporter ZIP10 for epidermal development: Implication of the ZIP10-p63 axis in epithelial homeostasis
Bin BH, Bhin J, Takaishi M, Toyoshima KE, Kawamata S, Ito K, Hara T, Watanabe T, Irié T, Takagishi T, Lee SH, Jung HS, Rho S, Seo J, Choi DH, Hwang D, Koseki H, Ohara O, Sano S, Tsuji T, Mishima K, Fukada TAbstract
Skin tissues, in particular the epidermis, are severely affected by zinc deficiency. However, the zinc-mediated mechanisms that maintain the cells that form the epidermis have not been established. Here, we report that the zinc transporter ZIP10 is highly expressed in the outer root sheath of hair follicles and plays critical roles in epidermal development. We found that ZIP10 marked epidermal progenitor cell subsets and that ablating Zip10 caused significant epidermal hypoplasia accompanied by down-regulation of the transactivation of p63, a master regulator of epidermal progenitor cell proliferation and differentiation. Both ZIP10 and p63 are significantly increased during epidermal development, in which ZIP10-mediated zinc influx promotes p63 transactivation. Collectively, these results indicate that ZIP10 plays important roles in epidermal development via, at least in part, the ZIP10-zinc-p63 signaling axis, thereby highlighting the physiological significance of zinc regulation in the maintenance of skin epidermis.Keywords
ZIP10; development; hair follicle; skin epidermis; zinc transporter
Anti-Pigmentary Effect of (-)-4-Hydroxysattabacin from the Marine-Derived Bacterium Bacillus sp
Kim K, Leutou AS, Jeong H, Kim D, Seong CN, Nam SJ, Lim KM.Abstract
Bioactivity-guided isolation of a crude extract from a culture broth of Bacillus sp. has led to the isolation of (-)-4-hydroxysattabacin (1). The inhibitory effect of (-)-4-hydroxysattabacin (1) was investigated on melanogenesis in the murine melanoma cell line, B16F10, and human melanoma cell line, MNT-1, as well as a pigmented 3D-human skin model. (-)-4-Hydroxysattabacin treatment decreased melanin contents in a dose-dependent manner in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. Quantitative real time PCR (qRT-PCR) demonstrated that treatment with (-)-4-hydroxysattabacin down-regulated several melanogenic genes, including tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) while their enzymatic activities were unaffected. The anti-melanogenic effects of (-)-4-hydroxysattabacin were further demonstrated in a pigmented 3D human epidermal skin model, MelanodermTM, and manifested as whitening and regression of melanocyte activation in the tissue.Keywords
(-)-4-hydroxysattabacin; Bacillus sp.; anti-pigmentary effect; marine-derived; melanogenesis
Exploration and comparison of in vitro eye irritation tests with the ISO standard in vivo rabbit test for the evaluation of the ocular irritancy of contact lenses.
Yun JW, Hailian Q, Na Y, Kang BC, Yoon JH, Cho EY, Lee M, Kim DE, Bae S, Seok SH, Lim KM.Abstract
In an effort to explore the use of alternative methods to animal testing for the evaluation of the ocular irritancy of medical devices, we evaluated representative contact lenses with the bovine corneal opacity and permeability test (BCOP) and an in vitro eye irritation test using the three-dimensionally-reconstructed human corneal epithelium (RhCE) models, EpiOcular™ and MCTT HCE™. In addition, we compared the obtained results with the ISO standard in vivo rabbit eye irritation test (ISO10993-10). Along with the positive controls (benzalkonium chloride, BAK, 0.02, 0.2, and 1%), the extracts of 4 representative contact lenses (soft, disposable, hard, and colored lenses) and 2 reference lenses (dye-eluting and BAK-coated lenses) were tested. All the lenses, except for the BAK-coated lens, were determined non-irritants in all test methods, while the positive controls yielded relevant results. More importantly, BCOP, EpiOcular™, and MCTT HCE™ yielded a consistent decision for all the tested samples, with the exception of 0.2% BAK in BCOP, for which no prediction could be made. Overall, all the in vitro tests correlated well with the in vivo rabbit eye irritation test, and furthermore, the combination of in vitro tests as a tiered testing strategy was able to produce results similar to those seen in vivo. These observations suggest that such methods can be used as alternative assays to replace the conventional in vivo test method in the evaluation of the ocular irritancy of ophthalmic medical devices, although further study is necessary.Keywords
Animal alternative; BCOP; Contact lens; Eye irritation; ISO10993-10; Medical device; Reconstructed human corneal epithelium, RhCE model
Evaluating the Micronucleus Induction Potential for the Genotoxicity Assay Using the Human Skin Model, KeraSkin™
Su-Hyon Lee, Haeng-Sun Jung, Seol-Yeong Kim, Hye Soo Kim, Kyung-Min Lim, Young-Shin Chung, and Tae-Boo ChoeAbstract
Micronucleus test is genotoxicity assay for detection of micronuclei in the cytoplasm of interphase cells. The reduction and replacement of in vivo toxicity testing on animals require the development of in vitro models to predict the genotoxicity or other tests for cosmetic products. In this study, we evaluated a genotoxicity assay for topically applied chemicals using a three-dimensional human reconstructed skin model, KeraSkinTM. Two genotoxins, mitomycin C (MMC) and methyl methanesulfonate (MMS), induced significant dose-related increases in cytotoxicity and micronuclei induction in the skin model. In contrast, two non-genotoxins, 4-nitrophenol (4-NP)and trichloroethylene (TCE), induced cytotoxicity but not micronucleus formation. In conclusion, micronucleus test using human skin model may be useful for predicting in vitro genotoxic potentials of cosmetic products.Keywords
reconstructed human epidermis, KeraSkinTM, genotoxicity, micronucleustest
An acrodermatitis enteropathica-associated Zn transporter, ZIP4, regulates human epidermal homeostasis
Bum-Ho Bin, Jinhyuk Bhin, Nan-Hyung Kim, Su-Hyon Lee, Haeng-Sun Jung, Juyeon Seo, Dae-Kyum Kim, Daehee Hwang, Toshiyuki Fukada, Ai-Young Lee, Tae Ryong LeeAbstract
Acrodermatitis enteropathica (AE) is an autosomal recessive disorder that is characterized by scaly eczematous dermatosis accompanied by alopecia and diarrhea. Various mutations in the SLC39A4 gene (ZIP4), which encodes a zinc transporter, are responsible for this disorder. However, the molecular mechanism underlying the involvement of ZIP4 in the pathogenesis of this condition has yet to be established. In this study, we report the role of ZIP4 in human epidermis. ZIP4 is predominantly expressed in human keratinocytes, and its expression is dramatically reduced upon epidermal differentiation. ZIP4 knockdown in human keratinocytes down-regulates Zn levels and the transcriptional activity of a key epidermal Zn-binding protein, ΔNp63, and dysregulates epidermal differentiation in a reconstituted human skin model, resulting in the appearance of proliferating keratinocytes even in the uppermost layers of the skin. We verified that, among the amino acid residues in its Zn-binding motif, Cys205 is critical for the processing and nuclear distribution of ΔNp63 and, therefore, Zn-dependent transcriptional activity. Our results suggest that ZIP4 is essential for maintaining human epidermal homeostasis through the regulation of Zn-dependent ΔNp63 activity and can provide insight into the molecular mechanisms responsible for the cutaneous symptoms observed in AE patients.Keywords
zinc; acrodermatitis enteropathica; zinc transporter; epidermis; p63Abbreviations
ZIP, Zrt- and Irt-like proteins; MT, metallothionein; AE, acrodermatitis enteropathica
Prevalidation trial for a novel in vitro eye irritation test using the reconstructed human cornea-like epithelial model, MCTT HCE™
Hyeri Yang, Da-eun Kim, Won-Hee Jang, Susun An, Sun-A Cho, Mi-Sook Jung, Ji Eun Lee, Kyung-Wook Yeo, Sang Bum Koh, Tae-Cheon Jeong, Mi-Jeong Kang, Young-Jin Chun, Su-Hyon Lee, Kyung-Min Lim, SeungJin BaeAbstract
Here, we report the results of a prevalidation trial for an in vitro eye irritation test (EIT) using the reconstructed human cornea-like epithelium, MCTT HCE™. The optimal cutoff to determine irritation in the prediction model was established at 35% with the receiver operation characteristics(ROC) curve for 126 substances. Within-lab(WL) and between-lab(BL) reproducibility was tested for 20 reference substances by 3 participating laboratories. Viability data described by mean ± SD or ± 1/2 difference between duplicate wells, and scatter plots, demonstrated the WL/BL consistency. WL/BL concordance with the binary decision, whether non-irritant or irritant was estimated to be 85–95% and 95%, respectively. WL/BL reproducibility of viability data was further supported by a strong correlation(ICC, r > 0.9). WL/BL agreement of binary decisions was also examined by Fleiss' Kappa statistics, which showed a strong level of agreement (> 0.78), nevertheless weaker than the reproducibility of the viability. The EIT with MCTT HCE™ exhibited a sensitivity of 82.2% (60/73), a specificity of 81.1% (43/53), and an accuracy of 81.8% (103/126) for 126 reference substances (for liquids; a sensitivity of 100% (47/47), a specificity of 70.6% (24/34), and an accuracy of 87.7% (71/81), and for solids, a sensitivity of 50% (13/26), a specificity of 100% (19/19), and an accuracy of 71.1% (32/45), suggesting that the accuracy is satisfactory but the sensitivity needs improvement, which shall be addressed through correcting the poor sensitivity for solid substances in future full validation trials.Abbreviations
EIT, eye irritation test; RhCE, reconstructed human cornea-like epithelium; OECD TG, OECD Test guidelines; GHS, global harmonization system; WL/BL, within-/between-laboratory; ROC, receiver operating characteristics; SD, standard deviation; AUC, area under curve; ICC, intra-class correlation; PS, performance standardKeywords
Eye irritation test; MCTT HCE™; Reproducibility; Predictive capacity; Statistical analysis
Evaluation of Eye Irritation Potential of Solid Substance with New 3D Reconstructed Human Cornea Model, MCTT HCE™
Won-hee Jang, Kyoung-mi Jung, Hye-ri Yang, Miri Lee, Haeng-Sun Jung, Su-Hyon Lee, Miyoung Park, and Kyung-Min LimAbstract
The eye irritation potential of drug candidates or pharmaceutical ingredients should be evaluated if there is a possibility of ocular exposure. Traditionally, the ocular irritation has been evaluated by the rabbit Draize test. However, rabbit eyes are more sensitive to irritants than human eyes, therefore substantial level of false positives are unavoidable. To resolve this species difference, several three-dimensional human corneal epithelial (HCE) models have been developed as alternative eye irritation test methods. Recently, we introduced a new HCE model, MCTT HCETM which is reconstructed with non-transformed human corneal cells from limbal tissues. Here, we examined if MCTT HCETM can be employed to evaluate eye irritation potential of solid substances. Through optimization of washing method and exposure time, treatment time was established as 10 min and washing procedure was set up as 4 times of washing with 10 mL of PBS and shaking in 30 mL of PBS in a beaker. With the established eye irritation test protocol, 11 solid substances (5 non-irritants, 6 irritants) were evaluated which demonstrated an excellent predictive capacity (100% accuracy, 100% specificity and 100% sensitivity). We also compared the performance of our test method with rabbit Draize test results and in vitro cytotoxicity test with 2D human corneal epithelial cell lines.Keywords
Eye irritation, Reconstructed cornea model, MCTT HCETM model, Protocol refinement
Skin corrosion and irritation test of sunscreen nanoparticles using reconstructed 3D human skin model
Jonghye Choi, Hyejin Kim, Jinhee Choi, Seung Min Oh, Jeonggue Park, Kwangsik ParkAbstract Objectives
Effects of nanoparticles including zinc oxide nanoparticles, titanium oxide nanoparticles, and their mixtures on skin corrosion and irritation were investigated by using in vitro 3D human skin models (KeraSkin™) and the results were compared to those of an in vivo animal test.Methods
Skin models were incubated with nanoparticles for a definite time period and cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide method. Skin corrosion and irritation were identified by the decreased viability based on the pre-determined threshold.Results
Cell viability after exposure to nanomaterial was not decreased to the pre-determined threshold level, which was 15% after 60 minutes exposure in corrosion test and 50% after 45 minutes exposure in the irritation test. IL-1α release and histopathological findings support the results of cell viability test. In vivo test using rabbits also showed non-corrosive and non-irritant results.Conclusions
The findings provide the evidence that zinc oxide nanoparticles, titanium oxide nanoparticles and their mixture are ‘non corrosive’ and ‘non-irritant’ to the human skin by a globally harmonized classification system. In vivo test using animals can be replaced by an alternative in vitro test.Keywords
Alternative methods, Corrosion, 3D skin model, Irritation, Nanoparticles
Identification of cornifelin and early growth response-1 gene as novel biomarkers for in vitro eye irritation using a 3D reconstructed human cornea model MCTT HCE™
Choi S, Lee M, Lee SH, Jung HS, Kim SY, Chung TY, Choe TB, Chun YJ, Lim KM.Abstract
Evaluation of the eye irritation is essential in the development of new cosmetic products. Draize rabbit eye irritation test has been widely used in which chemicals are directly applied to rabbit eye, and the symptoms and signs of eyes are scored. However, due to the invasive procedure, it causes substantial pain and discomfort to animals. Recently, we reported in vitro eye irritation test method using a 3D human corneal epithelial model (MCTT HCE™) which is reconstructed from remaining human tissues after a corneal transplantation. This model exhibited an excellent predictive capacity for 25 reference chemicals (sensitivity 100%, specificity 77% and accuracy 88% vs. GHS). To improve the test performance, we explored new biomarkers for the eye irritation through transcriptomic approach. Three surfactants were selected as model eye irritants that include sodium lauryl sulfate, benzalkonium chloride and triton X-100. After test chemicals were treated, we investigated differentially expressed genes through a whole-gene microarray (Affymetrix GeneChip(®) Human Gene 2.0 ST Array, 48,000 probes). As a result, we identified that mRNAs of cornifelin (CNFN), a constituent of the insoluble cornified cell envelope of stratified squamous epithelia, and early growth response-1 (EGR1), a nuclear transcriptional regulator, were significantly up-regulated by all three irritants. Up-regulation of CNFN and EGR1 was further confirmed by Q-RT-PCR, and immunohistochemistry revealed increased level of CNFN in irritant-treated tissues, supporting the relevance of CNFN and EGR1 as new biomarkers for eye irritation.
KeraSkin-VM: a novel reconstructed human epidermis model for skin irritation tests.
Jung KM, Lee SH, Jang WH, Jung HS, Heo Y, Park YH, Bae S, Lim KM, Seok SH.Abstract
Several alternative in vitro methods to evaluate skin irritants have been developed recently. In July 2010, OECD officially endorsed the validated reference method (VRM) that uses reconstituted human epidermis (RhE) models as replacements for the in vivo skin irritation test. This study evaluated the KeraSkin-VM model, a novel human epidermis model that was reconstructed with Asian skin tissue using 20 reference chemicals according to the OECD TG 439 performance standard. The test chemicals were applied to the epidermal surface side for 45 min and then rinsed, and then incubated for 42 h post-treatment. An overall accuracy of 80%, sensitivity of 90% and specificity of 70% were obtained when the results from KeraSkin-VM were compared with UN GHS categories, which was comparable to the EpiDerm Skin irritation test (SIT) rates. Furthermore, KeraSkin-VM demonstrated good performance in terms of within-laboratory reproducibility and predictive capacity to screen skin irritants.
Evaluation of permeability and irritancy of transdermal tranexamic acid formulation using a reconstructed human epidermis, Keraskin™
Yang-Hui Park, Won-Hee Jang, Kyoung-Mi Jung, Su-Hyun Lee, Kyung-Min LimAbstract
The skin permeability of active ingredient is one of the most important issues for designing transdermal administration form. At the same time, the formulation should be non-irritant to skin. To evaluate skin permeability and irritancy, various methods have been proposed and ex vivo skin is the tool recommended by regulators. However its use is time consuming and requires numerous human donors. Owing to the similarity to real skin, reconstructed human epidermis model have been recognized as a useful tool. We simultaneously evaluated the permeability and irritancy of tranexamic acid in transdermal formulation using the Keraskin™ of MCTT, a three-dimensional reconstructed human epidermis. We applied 4 transdermal formulations to the Keraskin™ and after incubation for 6 hr, culture medium was collected. Keraskin™ was washed five times with DPBS and MTT assay was performed for measuring cell viability. The collected medium was analyzed by HPLC-MS/MS to determine the tranexamic acid concentration. Cell viability was calculated through dividing with the absorbance of MTT formazan of AHA/BHA non-containing vehicle group. No significant differences were found in four groups. The concentration of tranexamic acid in culture medium was determined by HPLCMS/MS; 11.0 ± 2.5μg/mL in tranexamic acid in AHA/BHA non-containing formulation, 24.6 ± 3.8μg/mL in tranexamic acid in AHA/BHA containing formulation and not detected in vehicle groups. These findings demonstrated that reconstructed human epidermis can be used for the simultaneous evaluation of the skin permeability and irritancy of transdermal drug and cosmetic ingredients or products.Keywords
reconstructed human epidermis, permeability, irritancy, tranexamic acid
Evaluation of moisturizing activity of Lipid-coated powder using a reconstructed human epidermis
Won Hee Jang, Kyoung Mi Jung, Sun Kyung Choi, Haeng Sun Jung, Su Hyon Lee, Young Jin Choi, Young Ho Park, Kyung Min Lim.Abstract
Dry skin is induced by excessive transdermal water loss due to the damages of intracellular lipids. Moisturizer is safe and effective for improving dry skin, however, there is few efficient alternative method to evaluate moisturizing activity of new cosmetic ingredients. Here, we investigated the moisturizing activity of a face powder coated with mannosylerithritol lipids (MEL) that are similar to ceramides in structure and intermediates in the biosynthesis of intracellular lipids, without using animals or human but Keraskin™, a three-dimensional reconstructed human epidermis. After Keraskin™ was incubated, MEL-coated sericite or uncoated sericite was applied and then sodium lauryl sulphate (SLS) was treated with a nylon mesh covering to spread it uniformly. After incubation for 15 min, Keraskin™ was washed and it was post-incubated for 24 hr. At the end of post-incubation, Keraskin™ was incubated with MTT for 3 hr to measure viability. Formed purple formazan was extracted with isopropanol for 2 hr. Absorbance of extract(A590) was measured using spectrophotometer. Cell viability was calculated by dividing with the absorbance of negative control. The viability of reconstructed epidermis treated 10% SLS was determined to be 12.3 ± 4.87%. When SLS was co-treated with sericite or MEL-coated sericite, cell viability was increased to 23.7 ± 2.92% or 71.5 ± 20% (p<0.05), respectively. MEL-coated sericite inhibited SLS-induced cytotoxicity substantially in comparison with sericite alone. Based on these results, reconstructed human epidermis can be used for the evaluation of the efficacy of cosmetic ingredients or products.Keywords
econstructed human epidermis, moisturizing activity, mannosylerithritol Lipid
A new 3D reconstituted human corneal epithelium model as an alternative method for the eye irritation test.
Jung KM, Lee SH, Ryu YH, Jang WH, Jung HS, Han JH, Seok SH, Park JH, Son Y, Park YH, Lim KM.Abstract
Many efforts are being made to develop new alternative in vitro test methods for the eye irritation test. Here we report a new reconstructed human corneal epithelial model (MCTT HCE model) prepared from primary-cultured human limbal epithelial cells as a new alternative in vitro eye irritation test method. In histological and immunohistochemical observation, MCTT HCE model displayed a morphology and biomarker expressions similar to intact human cornea. Moreover, the barrier function was well preserved as measured by high transepithelial electrical resistance, effective time-50 for Triton X-100, and corneal thickness. To employ the model as a new alternative method for eye irritation test, protocol refinement was performed and optimum assay condition was determined including treatment time, treatment volume, post-incubation time and rinsing method. Using the refined protocol, 25 reference chemicals with known eye irritation potentials were tested. With the viability cut-off value at 50%, chemicals were classified to irritant or non-irritant. When compared with GHS classification, the MCTT HCE model showed the accuracy of 88%, sensitivity of 100% and specificity of 77%. These results suggest that the MCTT HCE model might be useful as a new alternative eye irritation test method.
Interlaboratory Validation of KeraSkin™ for Replacement of Skin Irritation Test Using Animal Model
Ji-young Moon, Su-Hyon Lee, Haeng-Sun Jung, Yang-Hwan Ryu, Kyoung-Mi Jung, Kyung-Min Lim, Cheol-Beom Park.Abstract
Reconstructed skin provides advantages to single cell culture testing and leads to promising results as shown by different validation studies. In this study we investigated the interlaboratory validation for the efficacy of “KeraskinTM” model. We conducted endpoint analysis including cell viability (MTT reduction) and IL-1α release. Eighteen item, which were ECVAM test meterial, were selected for this validation study. Results of MTT assay were not different between EPI-200 and KeraSkinTM, but IL-1α levels of some test items different between EPI-200 and KeraSkinTM. We concluded that this KeraSkinTM model is useful for alternative skin irritation test, although other tests should be conducted for many other chemicals in international validation study.Keywords
Skin Irritation, EPI-200, KeraSkin, Alternative Method
Establishment of In Vitro Nasal Epithelial Model for Alternative Toxicity Test
Su-Hyon Lee, Haeng-Sun Jung, Yang-Hwan Ryu, Seog-Kyun Mun, Yeoun-Sook Chun, Jae-Chan Kim, and Youngsook Son.Abstract
Although there are strong urge to restrict the animal experiments for toxicity test, the alternative in-vitro test model or guideline is not yet been established. The present study attempted to establish the in-vitro nasal epithelial model for alternative toxicity test. Human nasal epithelial(HNE) cells were isolated by dispase II treatment, and cultured monolayer until confluent. HNE cells were harvested and seeded onto 12 mm millicell. The cells were cultured for 7 days of submerged condition and following 7 days of air-liquid interface(ALI) condition. Fully differentiated cell sheets were prepared 0.4 μm sections, and H&E and immunohistochemical staining were compared with intact human nasal epithelium and conjunctiva. Primary cultured HNE cells showed cobblestone-like morphology without contamination with other cell types. Histological observation showed well differentiated ciliogenesis. The expressions of p63 at basal cell nucleus, CD44v6 at cell-cell junctions of basal cell layer, Na+/K+ ATPase at apical layer was observed. Also ColIV, LN5, CK3/12, CK13, CK5, MUC1, and MUC5AC were positively expressed.
These results suggest that in-vitro differentiated nasal epithelial model is suitable for alternative toxicity test due to its similarity with nasal epithelium. Taken together, our nasal epithelial model will be useful for alternative toxicity test.
Reconstructed nasal epithelium, 3D in-vitro model, alternative toxicity test
Efficacy of Reconstituted Human Corneal Model to Assess the Ocular Irritating Test
Haeng-Sun Jung, Su-Hyon Lee, Yang-Hwan Ryu, Kyoung-Mi Jung, Chae-Wook Kim, Kyung-Min Lim, Youngsook SonAbstract
Alternative methods to the Draize eye irritation test, such as the BCOP, HET-CAM, ICE, and IRE, are used to evaluate the ocular irritation potential of cosmetic, livelihood articles or industrial chemicals. In order to improve the sensitivity and specificity of alternative eye irritation test, we developed a three-dimensional human corneal model that uses normal human corneal epithelial cells. Our corneal tissue model consists of normal human corneal epithelial cells on cell culture inserts at the air liquid interface, differentiating to form a stratified, squamous epithelium similar to that found in the human cornea, exhibiting in vivo like morphological and histological characteristics. In this study, two laboratories have tested 20 reference chemicals using the same study protocol. The results were compared to in vivo data as well as previously published data obtained in the other three-dimensional corneal model test. Although an overall accuracy of 85% was obtained (sensitivity=100% and specificity=67%), further experiments are required to confirm and validate these preliminary results.Keywords
Eye irritation, Draize test, human corneal model, alternative method
The validation of alternative methods of reconstructed human skin equivalents for the assessment of skin irritation
Kyoung-Mi Jung, Ji-Young Moon, Su-Hyon Lee, Chae-Wook Kim, Cheol-Beom Park and Bae-Hwan KimAbstract
Several alternative in vitro methods for identifying skin irritants have been developed recently, the most promising of which use reconstituted human skin model. In this study, the two reconstituted human skin models, KeraskinTM and EPI-200, showed promising results in terms of interlaboratory variability and predictive capacity. The main endpoint measured for both KeraskinTM and EPI-200 was cell viability via MTT. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1α release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. In the interlaboratory validation study for KeraskinTM, sensitivity, specificity and accuracy of the KeraskinTM were 90%, 44% and 68%(MTT assay only), respectively. The KeraskinTM model was overly sensitive, resulting in the prediction of the non-irritant chemicals as irritant. The EPI-200 model had an accuracy of 63%, with a sensitivity rate of 70% and a specificity rate of 56%. Based on these results, although predictive capacity of KeraSkinTM model was considered insufficient, due to a low specificity, we concluded that this KeraSkinTM model is useful potential alternative skin irritation method. Further studies are in progress to improve the test protocols and prediction models for KeraSkinTM.Keywords
Skin irritation, KeraSkinTM, EPI-200, Alternative method
Efficacy of reconstituted skin for replacement of skin irritation test using animal model
Ji Young Moon, Eun Ho Maeng, Hak Soo Park, Min Kwon, Dong Hyouk Jang, Young Min Cho, Eun Ok Koh, Ha Jung Sung, Cheol Beom ParkAbstract
Reconstructed skin provides advantages to single cell culture testing and leads to promising results as shown by different validation studies. In this study we investigated the efficacy of "MCTT Skin model" compared with "EPI-200" and in vivo test. We conducted endpoint analysis including cell viability (MTT reduction, CCK-8) and IL-1 alpha release. Sodium lauryl sulfate(20%), Potassium hydoxide(5%), Heptanal, Methyl palmitate, 1,1,1-Trichloroethane, 2,4-Xylidine, 3,3`-Dithiodipropionic acid, 4-Amino-1,2,4-triazol and Benzyl chloroformate were selected for this comparison. Potassium hydoxide(5%) and Benzyl chloroformate were excluded from animal tests because of their pH. Sodium lauryl sulfate induced severe irritation, Heptanal induced moderate irritation. Methyl palmitate, 1,1,1-Trichloroethane induced mild irritation. 2,4-Xylidine, 3,3`-Dithiodipropionic acid, 4-Amino-1,2,4-triazole induced no irritation on rabbit skin irritation test. Results of MTT assay and CCK-8 assay were not different between EPI-200 MCTT Skin model but IL-lα levels of some test items different between EPI-200 and MCTT Skin model. We concluded that this MCTT Skin model is useful for alternative skin irritation test, although other tests should be conducted for many other chemicals in international validation study.Keywords
Skin irritation, EPI-200, MCTT Skin model, alternative method
Skin permeation, biodistribution, and expression of topically applied plasmid DNA.
Kang MJ, Kim CK, Kim MY, Hwang TS, Kang SY, Kim WK, Ko JJ, Oh YK.Abstract
BACKGROUND: Topical application is emerging as a new route of gene delivery. However, the extent of skin permeation and the in vivo fate of topically applied plasmid DNA are not fully understood.
METHODS: In vitro permeation of plasmid DNA across human skin and keratinocyte layers was tested using Franz diffusion cells. In vivo absorption and biodistribution of topically applied plasmid in mice were determined using quantitative polymerase chain reaction (PCR). The expression levels of plasmid DNA in various tissues were measured by semiquantitative reverse transcription PCR.
RESULTS: In vitro, topically applied DNA was capable of penetrating human skin and keratinocyte layers. Following topical application of plasmid DNA onto murine skin, the levels of plasmid DNA in the serum peaked at 4 hr. At 24 hr post-dose, topically applied DNA existed at higher levels than intravenously administered DNA in almost all tissues, and induced 11.4- and 22-fold higher mRNA expression in muscle and skin, respectively. Moreover, the topical route showed sustained expression of plasmid DNA in the regional lymph nodes over 5 days, whereas the intravenous route did not.
CONCLUSIONS: Taken together, our results show that topically applied plasmid DNA is capable of permeating the skin and being expressed for prolonged periods in various tissues including lymph nodes. This suggests that skin may provide an appealing, noninvasive route of delivery for DNA vaccines and other therapeutic genes.
Evaluation for oral mucosa irritation of alcohol concentration using 3D reconstructed human oral tissue model
Hye Soo Kim, Seol-Yeong Kim, Haeng-Sun Jung, Ji-Hae Chang, Sun Young Jo, Su-Hyon LeeAbstract
In order to keep healthy and hygienic teeth, various oral care products such as mouthwashes are used. A lot of oral care products on sale contain high level of alcohol to enhance a feeling of refreshment and remove bad breath. However, as the previous study in animal test or clinical trials, alcohol in oral care product may cause xerostamia and hypersensitive reaction. Thus, oral products containing high concentrations of alcohol are required safety confirmation. Meanwhile, EU cosmetics legislation forbids animal testing of cosmetics and its ingredient for animal welfare. Far more, the experiment with human oral cell line or human oral 3D tissue is used lately. The aim of this study is the evaluation for the implication of alcohol level in mouthrinses on oral mucosa using the human oral mucosa model. We estimated oral irritation of four types of mouthrinses of different alcohol level by conducting the MTT assay, cytokine ELISA and histological analysis. By the results, we could predict that there was little irritant potential of mouthrinses in the oral mucosa model. In addition, according to the alcohol concentration-dependent cell viability and production of pro-inflammatory cytokine such as IL-1alpha, we could suggest this system can evaluate the degree of marginal irritation. Therefore, the present study proposes that this new in vitro method by using human oral mucosa model could be used for evaluation of oral irritation to replace animal test.Keywords
Oral mucosa irritation, Human oral tissue model, Mouthrinses, Alcohol concentration
Assessment of safety and efficacy of cosmetic ingredients using reconstructed human epidermal model, KeraSkin™-VM
Hye Soo Kim, Seol-Yeong Kim, Haeng-Sun Jung, Ji-Hae Chang, Sun Young Jo, Su-Hyon LeeAbstract
Evaluation of safety and effectiveness of cosmetic or its ingredient has traditionally been conducted in animal models. However, a growing focus and consensus for minimizing animal use have stimulated the development of alternative in vitro methods such as using cell lines or human 3D skin tissue model. KeraSkin, reconstructed human tissue model, reflects metabolically complexities of in vivo as well as human specific responses. Therefore, the aim of this study is investigation of novel in vitro test systems for the implication of the safety and effect of cosmetic raw materials by using KeraSkin. To assess the safety of cosmetic raw material such as STem水, which consists of conditioned media of adipose-derived stem cell using KeraSkin, it was applied topically on KeraSkin at increasing concentrations and measured cell viability by MTT assay according to OECD TG439 which is the test guideline described an in vitro skin irritation test method using the reconstructed human epidermis. In addition, in order to determine the efficacy in the skin barrier functions as cosmetic raw materials, we prepared the skin model disrupted by SDS treatment. Following application of the cosmetic ingredients on the skin model, we evaluated skin barrier function via TEWL and immunohistochemistry. These results showed that KeraSkin can be used an alternative model to replace animal models for safety and efficacy testing for cosmetic raw materials.Keywords
KeraSkin™-VM, Cosmetic ingredients, OECD TG439, Skin irritation, Skin barrier function
The effect of conditioned media of adipose derived stem cells, STeM水, on the skin barrier function in reconstructed human epidermis model KeraSkin™-VM
Su-Hyon Lee, Haeng-SunJung, Seol-YeongKim, Ji-HaeChang , Jae-UnJungAbstract
Different from ordinary cells, stem cell is known to secrete cell growth factors beneficial to the growth and regeneration of skin, antioxidant substances, substances with moisturizing and brightening effects and unknown effective substances. Stem cell culture solution also contains such effective components. SteM 水 is a stem cell culture solution used as a cosmetic and is a core substance containing various growth factors developed through over five years’ research by key researchers of MCTT Co., Ltd., advanced biotechnology R&D company. This solution is best for the skin and provides the optimal environment and nutrition for the growth of stem cells. In order to evaluate the effects of SteM 水 on skin barrier function in vitro, KeraSkinTM-VM was used. KeraSkinTM-VM is reconstructed human tissue model, reflect metabolically complexities of in vivo and human specific responses and is used in safety or efficacy screening tests. In this study, KeraSkinTM-VM was exposed to SDS, and SteM水 was treated. Transepidermal water loss (TEWL) and epidermal expression markers were analyzed. This results showed that an STeM水 markedly prevented the SDS-induced increase in TEWL. The SDS-induced decrease in epidermal markers of proliferation and differentiation was also restored significantly by STeM水 treatment. This results reflected the notion that STeM水 can induce epidermal keratinocyte differentiation and can improve the recovery of skin barrier functions.Keywords
conditioned media, SteM水, skin model, KeraSkinTM, barrier function
Alternative in-vitro phototoxicity test using reconstructed skin model, KeraSkin™-VM
The reconstructed human skin model KeraSkin™-VM has similar morphology, characteristics, and even biochemical marker expressions to native human skin. This model had been showed usefulness as an alternative testing model in skin irritation and corrosion test. Although there are strong urge to restrict the animal experiments for toxicity test, the alternative in-vitro phototoxicity tests using KeraSkinTM-VM is not yet been established. Therefore, this study was conducted to validate the in-vitro phototoxicity test method using KeraSkin™-VM. Nine of phototoxic or non-phototoxic chemicals were topically treated onto KeraSkinTM-VM, and after 24 hrs incubation, the KeraSkin™-VM were exposed to 6 J/cm2 of UVA. Test chemicals were removed, and cell viability was quantified by MTT assay after incubation for another 24 hrs. Predictions of phototoxic potentials were highly reproduced, and the established prediction standard was effective showing consistency with previously reported in-vivo test results. In conclusion, in-vitro alternative phototoxicity test method using KeraSkinTM-VM was successfully established. Surely KeraSkin™-VM can be used as a good alternative test method for assessment of phototoxicity of the chemicals.
Evaluation of beneficial and adverse effects of glucocorticoids using a KeraSkinTM human skin model
Su-Hyon Lee, Haeng-Sun Jung, Seol-Yeong Kim, Ji-Hae Chang , Jae-Un JungAbstract
Glucocorticoids(GCs) are highly effective for the therapy of inflammatory skin disease. However their use is limited by their side effect potential, with skin thinning. Thus, determining the atrophogenic potential of novel compounds is of importance for drug development. Currently, there are no according predictive in vitro models available. Reconstructed human skin models present various advantages structurally and functionally as compared to mouse skin for human risk assessment. KeraskinTM, reconstructed human tissue model, reflect metabolically complexities of in vivo and human specific responses and is used in safety or efficacy screening tests. In this study, commercial GCs ointment were applied KeraskinTM model and evaluated anti-inflammation and atropy.
Development of new reconstituted human corneal model to assess the ocular irritating test
Su-Hyon Lee, Haeng-Sun Jung, Seol-Yeong Kim, Ji-Hae Chang, Jae-Eon Jung
R&D Institute, MCTT Bio, Inc., Seoul, Korea
Alternative methods to the Draize eye irritation test, such as the BCOP, HET-CAM, ICE, and IRE, are used to evaluate the ocular irritation potential of cosmetic, livelihood articles or industrial chemicals. In oreder to improve the sensitivity and specificity of alternative eye irriation test, we developed a novel three-dimentional human corenal model that uses a normal human corneal model that uses the normal human corneal epithelial cells. In this study, two laboratories have tested 20 reference chemicals using the same study protocol. The results were compared to previously published in vivo eye irritation as well as existing data obtained in the other three-dimensional corneal model test. A good intra/inter-laboratory reproducibility and correalstion with in vivo and other in vitro model results were obtained. Our new three-dimensional model, developed from normal human corneal epithelial cells, is reproducible and is accurately predicting model of eye irritation test.
Development of a New Reconstituted Human Oral Mucosal Model to Assess the Oral Irritation Testing
Su-Hyon Lee1, Haeng-Sun Jung1, Seol-Yeong Kim1, Song-Yi Yang2, Kwang-Mahn Kim2, Jae-Un Jung 1
1 R&D Institute, Modern Cell & Tissue Technologies, Inc., Seoul, Korea
2 Yonsei University College of Dentistry , Seoul, Korea
In this study we developed a novel three-dimensional human oral mucosal model (HOM model) based on two different cell sources. One is produced from immortalized human oral keratinocyte cell line (HOM-IHOK model) and the other is human normal buccal keratinocytes (HOM-NBK model). To compare these two oral mucosal model, immunohistochemistry and epidermal barrier properties were examined.
Development and characterization of Human MSC-derived dopaminergic neurons for drug discovery
Su-Hyon Lee1, Haeng-Sun Jung1, Seol-Yeong Kim1, Ji-Hae Chang1, Jae-Un Jung 1
1 R&D Institute, Modern Cell & Tissue Technologies, Inc., Seoul, Korea
Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into dopaminergic neurons, which is one of the major cell types damaged in Parkinson’s disease. In this study we differentiated hMSC into dopaminergic neurons and tested neuronal toxicity using a differentiated neural cells. The majority of the resulting cells displays typical neuronal morphology, with long and branching processes, and expresses key markers, including tyrosine hydroxylase (TH), MAP2 and Nurr1. Four neurotoxic substances were tested in neuronal cytotoxicity. This results provide dopaminergic neurons derived from hMSC will be available in neurotoxicity test.
Current researches in alternatives to animal testing
Hye Soo Kim, Su-Hyon Lee, Haeng-Sun Jung, Seol-Yeong Kim, Ji-Hae Chang, Jae-Eon Jung†
R&D Institute, MCTT Bio, Inc., Seoul, Korea
사람에게 사용되는 모든 제품들 중에 특히 화장품과 의약품은 인체에 직접 접촉하게 되므로 그에 대한 적절한 안전성이 확보되어야한다. 따라서 이러한 제품들은 개발단계에서 동물에게 먼저 시험하여 독성이 나타나지 않는다는 것을 먼저 확인하고 나서 사람에게 임상시험을 실시하여 최종적으로 안전성과 유효성을 확인하는 절차를 반드시 거친다. 이러한 안전성시험 과정은 많은 동물의 희생이 필요하므로 항상 동물의 생명 존중이라는 윤리적인 문제가 발생할 뿐만 아니라 동물에서 안전하다고 판정되었지만 사람에게는 안전하지 않게 나타날 수 있다는 문제점때문에 동물실험을 대체할 수 있는 시험법들이 개발되고 이를 공인된 가이드라인으로 제정하고자 하는 움직임이 활발히 일어나고 있다.
국제적으로 2003년 초 유럽연합(EU)에서 사람에게 사용하는 화장품의 안전성 평가를 위하여 실험동물사용을 금지하는 법안이 통과된 이후 2009년부터는 화장품원료까지 확장하여 급성 독성 실험에 동물사용을 완전히 금지하였으며 2013년 3월에 이르러서는 화장품 개발 전 과정에서 동물실험을 완전히 금지하도록 제정하였다. 이에 따라 특히 화장품분야에서는 in vitro test 방법을 개발하기 위하여 많은 연구가 진행되어 왔으며 현재 인공피부모델와 인공각막모델으로 독성 평가와 효능평가를 실시하여 화장품개발에 활용하는 단계에 이르렀다. 최근에는 동물실험 대체법을 연구하는 회사 및 연구소에서 피부감작, 피부자극성 및 부식성, 광독성, 안자극성 및 부식성시험 등을 포함하는 OECD 동물대체시험법 guideline의 평가항목에 다양한 인공조직 모델을 활용한 시험법을 등재시켜 사실상의 표준(de facto standard)을 선점하기 위한 경쟁이 치열하게 진행되고 있다.
사람에게서 유래한 세포를 이용하는 사람의 인공조직모델은 동물실험에 따르는 윤리적인 문제를 해결할 수 있을 뿐만 아니라, 실제 사람의 조직과 매우 유사하여 사람을 대상으로 하는 임상시험과 매우 유사한 결과를 기대할 수 있으며, 기존의 동물실험법보다 경제적이라는 장점 때문에 동물실험의 훌륭한 대안이 될 수 있다고 예상되고 있다.